I am due to start IHC on cerebellar organotypic slice culture (DIV21), and have some questions:
1. What is the recommended time frame for fixation? I saw, in different protocols, a range between 5min to 1h in RT.
2. What is the best possible way to remove the slices from the insert, when and where to? (e.g. petri dish? slide?)
3. I see now that the slices are covered with cells on top of them (glia?), Is it going to serve a physical barrier while staining?
Many thanks in advance!
Orli
Hallo Orli,
I am working on hippocampal slices, it think the steps are quite similar. We do the fixation for 30 minutes-1 hour at least. Sometimes it can be done over night.
We cut out the slices from the insert using a scalpel and then they are transferred into a new well plate with blockingsolution e.g. and in this wells we perform the staining as well and at the end we put them on slides for microscopic analysis.
Greetings =)
Hi Orli,
We have a couple of years of experience with organotypic cerebellar slice cultures in our lab. That are my answers:
1) We fix the slices in 4% PFA at RT or at 4°C for a couple of days
2) You can remove the slices from the insert as proposed by Orli. However, the membrane easily roll. Thus, as an alternative you can make holes with a needle into the membrane and perform the staining in a 6 well culture dish. Then the membranes are easily to handle. You remove the ring just at the end before you mount them.
3) Yes, on top an astrocytic glial layer start to grow as a reaction. Also as the slices are thicker than histological sections, incubate the first and second antibodies over several days (2-3).
Best,
Regina
Thank you Regina, It is very helpful, have you published any paper with the organotypic cerebellar slice culture protocol and IHC?
As far as I remember, the organotypic slices can easily be detached from the membrane after fixation for 3h at RT using a fine paintbrush. The slices can then be prepared to be frozen and cut at a microtome prior to putting them on a microscope slide and do the IHC protocol. Cutting to thinner sections will improve accessibility of your antibodies and improve image quality. However, if you are interested to keep the whole structure, probably you need to optimize permeabilization and incubation times as suggested by Regina.
Best,
Simon
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